Despite recent advances, cytokinesis remains the least understood aspect of the cell cycle. In animal cells, cytokinesis requires the coordination of multiple cellular components including the microtubule cytoskeleton, the actin cytoskeleton and membrane traffic. Today, it is not clear how these components are regulated and how they interact with each other. The long-term goal of this project is to define the elements involved in cytokinesis and to understand how these elements integrate to accomplish cytokinesis. Toward that goal, three proteins required for cytokinesis in Dictyostelium have been identified. These three proteins provide a handle on three different pathways that come together during cytokinesis. The objective of this grant is to dissect the contribution of these three pathways during cytokinesis: 1) The role of LvsA in cytokinesis and osmore, qulation will be determined. LvsA is a member of the novel beach family of membrane trafficking proteins and is required for cytokinesis and for the function of the contractile vacuole. This provides the opportunity to dissect the contribution of membrane traffic to cytokinesis in a simple model system. The functional contribution of each of the LvsA domains to its localization and function in vivo will be ascertained. Binding partners for LvsA will be identified and their requirement for cytokinesis will be tested. Genetic suppressors of LvsA will be isolated and the mechanism of suppression will be determined. These studies will illuminate our understanding of membrane processes in cytokinesis and also how mutations in a human homologue of LvsA (LYST) cause a human disorder, the Chediak-Higashi Syndrome. 2) The signaling pathway mediated by RacE and essential for cytokinesis will be delineated. The small GTPase Race is essential for the development of cortical tension and for the ingression of the cleavage furrow. A combination of approaches will be used to dissect the mechanisms by which racE controls cytokinesis and cortical tension. The hypothesis that racE promotes crosslinking of the actin cytoskeleton to enhance cortical tension will be tested. The requirement of cortical tension for the proper ingression of the cleavage furrow will be determined. Binding partners racE will be identified and their role in the development of cortical tension and cytokinesis will be determined. 3) The role of DdlNCENP in cytokinesis will be dissected. INCENP is a microtubule-binding protein known to be important for mitosis & cytokinesis in all organisms but its exact role in cytokinesis has not been identified. Our group has identified the Dictyostelium INCENP homologue and created a DdlNCENP knockout mutant. These tools will be used to explore the relationship of the microtubule cytoskeleton with the organization of the contractile ring during cytokinesis. Since INCENPs are known to be involved in tumorigenesis these results may provide insights into the mechanisms of tumor formation.